Human fibroblasts in culture as well as other types of human cells have proven to be extraordinarily difficult to transform into malignant cells. To study the mechanisms of cell transformation, we have developed the MSU-1 lineage. We began with normal human skin fibroblasts and by sequential, clonal selection, have now obtained more than 100 fully malignant cell strains using oncogene transfection or carcinogen treatment, and selection. One step that involves the spontaneous conversion of a telomerase positive, diploid, infinite life span strain, called MSU-1.0, to a telomerase positive, chromosomally stable, infinite life span strain called MSU-1.1 remains a black box. This step is very important because the MSU-1.1 cells can be readily transformed into malignant cells, but we have not been able to malignantly transform the MSU-I.0 cells. To identify differences in gene expression between these strains, we have carried out Affymetrix Gene Chip analysis. A total of 46 genes are expressed at a level at least 3-fold higher in the MSU-I.1 cells than in the MSU-1.0 cells; 21 genes are expressed at a level at least 3-fold lower in the MSU-1.1 cells than in the MSU-1.0 cells. To test the hypothesis that critical changes in gene expression in the MSU-1.1 cells are responsible for the fact that they can be malignantly transformed, we will: 1) confirm, by Northern blotting the differences between MSU-I.0 cells and MSU-I.1 cells in expression of each gene identified and if possible, by Western blotting, 2) determine whether genes whose expression were confirmed in 1 play a critical role in making the MSU-I.1 cells transformable by introducing by infection the cDNA of the genes of interest into the appropriate strain (i.e., genes expressed at a higher level in MSU-1.1 cells (Group 1) into MSU-1.0 cells and genes expressed at a lower level in MSU-1.1 cells(Group 2)into MSU-I.1 cells, by examining the cells' ability to grow in medium where growth factors are limiting and/or form colonies in agarose. Infected MSU-1.0 cells will be tested for their ability to grow under these conditions, infected MSU-1.1 cells for a reduction or blockage in cell growth. 3) determine whether the cell strains found to exhibit the appropriate critical change in growth factor responsiveness can be malignantly transformed by transfection of an H-RAS oncogene.